Niu Tian-Cai, Lin Gui-Ming, Xie Li-Rui, Wang Zi-Qian, Xing Wei-Yue, Zhang Ju-Yuan, Zhang Cheng-Cai
Key Laboratory of Algal Biology , Institute of Hydrobiology, the Chinese Academy of Sciences , Wuhan , Hubei Province , China.
University of Chinese Academy of Sciences , Beijing , 100049 , China.
ACS Synth Biol. 2019 Jan 18;8(1):170-180. doi: 10.1021/acssynbio.8b00437. Epub 2018 Dec 21.
CRISPR systems, such as CRISPR-Cas9 and CRISPR-Cpf1, have been successfully used for genome editing in a variety of organisms. Although the technique of CRISPR-Cpf1 has been applied in cyanobacteria recently, its use was limited without exploiting the full potential of such a powerful genetic system. Using the cyanobacterium Anabaena PCC 7120 as a model strain, we improved the tools and designed genetic strategies based on CRISPR-Cpf1, which enabled us to realize genetic experiments that have been so far difficult to do in cyanobacteria. The development includes: (1) a "two-spacers" strategy for single genomic modification, with a success rate close to 100%; (2) rapid multiple genome editing using editing plasmids with different resistance markers; (3) using sacB, a counter-selection marker conferring sucrose sensitivity, to enable the active loss of the editing plasmids and facilitate multiple rounds of genetic modification or phenotypic analysis; (4) manipulation of essential genes by the creation of conditional mutants, using as example, polA encoding the DNA polymerase I essential for DNA replication and repair; (5) large DNA fragment deletion, up to 118 kb, from the Anabaena chromosome, corresponding to the largest bacterial chromosomal region removed with CRISPR systems so far. The genome editing vectors and the strategies developed here will expand our ability to study and engineer cyanobacteria, which are extensively used for fundamental studies, biotechnological applications including biofuel production, and synthetic biology research. The vectors developed here have a broad host range, and could be readily used for genetic modification in other microorganisms.
CRISPR系统，如CRISPR-Cas9和CRISPR-Cpf1，已成功用于多种生物体的基因组编辑。尽管CRISPR-Cpf1技术最近已应用于蓝细菌，但在未充分发挥这种强大遗传系统潜力的情况下，其应用受到限制。以蓝细菌鱼腥藻PCC 7120为模式菌株，我们改进了工具并设计了基于CRISPR-Cpf1的遗传策略，这使我们能够实现迄今为止在蓝细菌中难以开展的遗传实验。进展包括：（1）用于单基因组修饰的“双间隔序列”策略，成功率接近100%；（2）使用具有不同抗性标记的编辑质粒进行快速多基因组编辑；（3）使用赋予蔗糖敏感性的反选择标记sacB，使编辑质粒主动丢失，并便于进行多轮遗传修饰或表型分析；（4）通过创建条件突变体来操纵必需基因，例如以编码DNA复制和修复所必需的DNA聚合酶I的polA为例；（5）从鱼腥藻染色体上删除长达118 kb的大片段DNA，这是迄今为止用CRISPR系统去除的最大细菌染色体区域。本文开发的基因组编辑载体和策略将扩展我们研究和改造蓝细菌的能力，蓝细菌广泛用于基础研究、包括生物燃料生产在内的生物技术应用以及合成生物学研究。本文开发的载体具有广泛的宿主范围，可轻易用于其他微生物的遗传修饰。
